Bisindolylmaleimide I Research Articles

Formulation of Liver-Specific PLGA-DY-635 Nanoparticles Loaded with the Protein Kinase C Inhibitor Bisindolylmaleimide I.

Bisindolylmaleimide I (BIM-I) is a competitive pan protein kinase C inhibitor with anti-inflammatory and anti-metastatic properties, suggested to treat inflammatory diseases and various cancer entities. However, despite its therapeutic potential, BIM-I has two major drawbacks, i.e., it has a poor water solubility, and it binds the human ether-à-go-go-related gene (hERG) ion channels, potentially causing deadly arrhythmias. In this case, a targeted delivery of BIM-I is imperative to minimize peripheral side effects. To circumvent these drawbacks BIM-I was encapsulated into nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) functionalized by the near-infrared dye DY-635. DY-635 served as an active targeting moiety since it selectively binds the OATP1B1 and OATP1B3 transporters that are highly expressed in liver and cancer cells. PLGA-DY-635 (BIM-I) nanoparticles were produced by nanoprecipitation and characterized using dynamic light scattering, analytical ultracentrifugation, and cryogenic transmission electron microscopy. Particle sizes were found to be in the range of 20 to 70 nm, while a difference in sizes between the drug-loaded and unloaded particles was observed by all analytical techniques. In vitro studies demonstrated that PLGA-DY-635 (BIM-I) NPs prevent the PKC activation efficiently, proving the efficacy of the inhibitor after its encapsulation, and suggesting that BIM-I is released from the PLGA-NPs. Ultimately, our results present a feasible formulation strategy that improved the cytotoxicity profile of BIM-I and showed a high cellular uptake in the liver as demonstrated in vivo by intravital microscopy investigations.

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca(2+) signaling must be tightly controlled. Thrombin-induced Ca(2+) signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca(2+) signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. To investigate the mechanisms by which PKC inhibitors enhance thrombin-induced Ca(2+) signaling, and to determine the importance of this pathway in platelet activation. Cytosolic Ca(2+) signaling was monitored in fura-2-loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. PKC inhibition by bisindolylmaleimide-I (BIM-I) enhanced α-thrombin-induced Ca(2+) signaling in a concentration-dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ-thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM-I had little effect on α-thrombin-induced signaling following treatment with the PAR1 antagonist, SCH-79797. BIM-I enhanced Ca(2+) release from intracellular stores and Ca(2+) entry, as assessed by Mn(2+) quench. However, the plasma membrane Ca(2+) ATPase inhibitor, 5(6)-carboxyeosin, did not prevent the effect of BIM-I. PKC inhibition strongly enhanced α-thrombin-induced PS exposure, which was reversed by blockade of PAR1. Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca(2+) release and Ca(2+) entry, which leads to reduced platelet PS exposure.

A complex of Ca V 1.2/PKC is involved in muscarinic signaling in smooth muscle

Here we present functional and biochemical evidence for a Ca(2+) channel (Ca(V)1.2)/protein kinase C (PKC) signaling complex being a key player in muscarinic regulation of urinary bladder smooth muscle. Muscarinic stimulation induced Ca(2+) signals and concomitant contractions in detrusor muscle from mice that were dependent on functional Ca(2+) channels. These signals were still present in muscles being depolarized by 85 mM extracellular K(+). Muscarinic-induced contractions were reduced by a PKC inhibitor [bisindolylmaleimide I (BIM-I)] and a phospholipase D (PLD) inhibitor (1-butanol). A phorbol ester (PDBu) enlarged muscarinic-induced Ca(2+) signals and contractions. The effects of BIM-I and PDBu were inhibited by isradipine and/or absent in muscles from Ca(V)1.2-deficient mice. Both carbachol and PDBu increased Ca(V)1.2 channel currents in isolated bladder myocytes. Blue native-PAGE electrophoresis revealed that Ca(V)1.2, PKC, and PLD are closely associated in muscles being previously stimulated by carbachol. Immunoprecipitation using anti-Ca(V)1.2 followed by Western blotting demonstrated that Ca(V)1.2 and PKC are coupled in stimulated muscles from wild-type mice. Autoradiography on immunoprecipitates showed that Ca(V)1.2 is a substrate for PKC-mediated phosphorylation. These findings suggest that a signaling complex consisting of Ca(V)1.2, PKC, and, probably, PLD controls muscarinic-mediated phasic contraction of urinary bladder smooth muscle.

Rapid Modulation of the Organic Anion Transporting Polypeptide 2B1 (OATP2B1, SLCO2B1) Function by Protein Kinase C-mediated Internalization

Members of the organic anion transporting polypeptide (OATP) family are involved in various pharmacological, pathophysiological, and physiological processes, such as hepatic drug uptake, progress of cancer, or transport of hormones. Although variability in expression and function of OATPs has been investigated in detail, data concerning regulation are rather limited. Here, we report a novel mechanism for rapid regulation of OATP2B1 mediated by protein kinase C (PKC) resulting in significant changes of transport activity. PKC activation by the phorbol ester (phorbol 12-myristate 13-acetate, PMA) resulted in increased phosphorylation of OATP2B1 as well as reduced OATP2B1 transport activity with a decrease in V(max) of E(1)S uptake (288 +/- 21 (control) versus 165 +/- 16 pmol/min/mg of protein (PMA)). This effect was sensitive to the PKC inhibitor bisindolylmaleimide I (BIM-I). Confocal microscopy, fluorescence-based internalization assay, and live-cell imaging using green fluorescent protein-tagged OATP2B1 revealed that transport inhibition was due to internalization of the transporter. Furthermore, colocalization with LAMP-2 and chloroquine-sensitive degradation of OATP2B1 suggest that the internalized protein is targeted to a lysosomal degradation pathway. With regard to the underlying mechanism inhibition of caveolin/lipid raft-mediated endocytosis failed to prevent OATP2B1 internalization, whereas inhibition of clathrin-mediated processes blocked OATP2B1 sequestration. However, small interfering RNA-mediated clathrin knock-down affected general trafficking of OATP2B1 and resulted in intracellular accumulation in the absence of PMA. In conclusion, our data demonstrate that OATP2B1 function is regulated by PKC-mediated, clathrin-dependent internalization and followed by lysosomal degradation. Furthermore, internalization could be shown in an ex vivo placenta perfusion. Our findings represent a new, rapid mechanism in regulation of human OATPs.

Eukaryotic ribosomes host PKC activity

PKC isoform βII modulates translation and can be recruited on ribosomes via its scaffold RACK1 (receptor for activated protein kinase C 1), which resides on the 40S ribosomal subunit. However, whether a PKC activity exists on the ribosome is not yet demonstrated. We purified native ribosomes by two different techniques, which avoid stripping of initiation factors and other associated proteins. In both cases, purified ribosomes are able to phosphorylate a specific PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate). MARCKS phosphorylation is switched on by treatment with PKC agonist PMA (Phorbol 12-Myristate 13-Acetate). Consistently, the broad PKC inhibitor BMI (Bisindolyl Maleimide I) abrogates MARCKS phosphorylation. These data show that native ribosomes host active PKC and hence allow the phosphorylation of ribosome-associated substrates like initiation factors and mRNA binding proteins.

EGF‐like peptides mediate FSH‐induced maturation of cumulus cell‐enclosed mouse oocytes

This study was carried out to examine the participation of epidermal growth factor (EGF)-like peptides in the induction of germinal vesicle breakdown (GVB) in mouse cumulus cell-enclosed oocytes (CEO). The EGF-like peptide, amphiregulin (AR), dose-dependently stimulated meiotic resumption in CEO, but not denuded oocytes (DO) maintained in meiotic arrest with 300 microM dbcAMP. The EGF receptor (EGFR) kinase inhibitor, AG1478, blocked meiotic resumption induced by FSH and AR in CEO, but had no effect in DO. FSH-induced maturation was also suppressed by antisera to both EGFR and EGF. Maturation occurred with slightly faster kinetics in AR-stimulated CEO when compared to FSH-stimulated CEO. When CEO were maintained in meiotic arrest with a low level of dbcAMP, FSH was initially inhibitory to maturation and later stimulatory; the stimulatory phase was prevented by AG1478, indicating mediation by EGF-like peptides. Pulsing CEO with high levels of dbcAMP also stimulated GVB and could be blocked by AG1478. Treatment of arrested CEO with PKC agonists stimulated maturation and this was prevented with AG1478 as well as antibodies to EGFR. FSH-induced maturation of dbcAMP-arrested CEO was blocked by bisindolylmaleimide I (BIM-I), an inhibitor of PKC, implicating PKC in FSH action. EGF-stimulated CEO failed to resume maturation in the presence of glycerrhetinic acid, a gap junction inhibitor, suggesting transfer of positive signal through the cell-cell coupling pathway. These data support the idea that EGF-like peptides provide a common pathway mediating the meiosis-inducing influence of FSH, cAMP pulsing, and PKC activation in mouse CEO by a gap junction-dependent process.

Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: The distinct role of protein kinase C and mitochondrial superoxide production

In this study the effects of stable and intermittent high glucose concentrations on ICAM-1, VCAM-1 and E-selectin production, PKC activity and PKCβI, βII and δ isoforms expression in cultured HUVEC have been examined. In stable high glucose ICAM-1, VCAM-1 and E-selectin concentration and mRNA expression increased, and this effect was even more evident in intermittent high glucose. PKC activity increased in fluctuating glucose compared to stable high glucose, due to an over-expression of βI, βII and δ isoforms. ICAM-1, VCAM-1 and E-selectin, after the adding of total PKC inhibitor bisindolylmaleimide-I (BIMI-I) and LY379196, a specific inhibitor of PKCβ, were equally reduced. 8-Hydroxydeoxyguanosine (8-OHdG), a sensitive indicator of oxidative damage to DNA, increased in stable and even more in intermittent high glucose and was reduced by both BIMI-I and LY379196. However, when thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial complex II and the SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were added, all adhesion molecules, any PKC isoforms expression and 8-hydroxydeoxyguanosine were normalized in both constant and oscillating glucose. In conclusion intermittent high glucose induces a greater expression of the adhesion molecules than stable high glucose; this effect seems to be related to an activation of PKCβ, but completely dependent from mitochondrial free radicals over-production.

Combined effects of protein kinase inhibitors and 5-fluorouracil on CEA expression in human colon cancer cells

Previous studies showed that 5-fluorouracil (5-FU) and Staurosporine (ST), a protein kinase inhibitor (PKI), were able to increase the expression of carcinoembryonic antigen (CEA) in human colon cancer cells. In the present study, we examined the in vitro effects of five PKIs, i.e. ST, 1-5-isoquinolinyl-sulfonyl-2-methylpiperazine (H-7), bisindolylmaleimide-I (BIS), Genistein (GEN), and Herbimycin A (HERB) alone or in combination with 5-FU on CEA expression. C22-20, a clonal subline, derived from colon cancer HT-29 line, selected for low expression of CEA, was used in our experimental model. Among the PKIs tested, only ST, at non-toxic concentrations of 5 nM, was capable of increasing the level of CEA. The other PKIs did not modify CEA expression when used either alone or in combination with 5-FU. Flow cytometric analysis showed that treatment of cells with 5-FU + ST resulted in a synergistic increase of CEA expression, being higher than that obtainable with both agents alone. Moreover, the increase of CEA expression occurred not only in membrane fractions but also in cytosolic compartments, as indicated by Western blot analysis. The present study suggests that ST-mediated induction of CEA expression in cancer cells is PKC independent and could be of potential clinical interest for the development of new diagnostic and/or immunotherapeutic approaches.

Effects of hypoxia on endothelial nitric oxide synthase gene expression in pulmonary artery endothelial cells and the role of protein kinase C isoforms

To explore the regulation of eNOS gene expression in pulmonary arterial endothelial cells (PAECs) by protein kinase C (PKC) and its isoforms during hypoxia. Primary cultured porcine PAECs were exposed to 5%O(2) for 2, 6, 12, 24, 48 hours. The eNOS mRNA level was measured by RT-PCR. Western blot technology was used to detect the contents of eNOS protein and the 8 PKC isoforms. After addition of selective PKC inhibitors, bisindolylmaleimideI (BIMI, 1 micro mol/L) or Gö6983 (1 micro mol/L), PAECs were exposed to 5%O(2) for 24 hours, then the expression of eNOS mRNA was detected by RT-PCR. Promoter activity of eNOS gene was determined by luciferase reporter gene assay. PAECs were transfected transiently with 1.6 kb fragment of the human eNOS promoter driving a luciferaes reporter gene, then exposed to 5%O(2). 24 h later, the activity of luciferase and beta-galactosidase was examined and the relative luciferase activity, representing the eNOS promotor activity, was calculated. After addition of actinomycine D (5 micro g/ml) and exposure to 5%O(2) or normoxia for 6, 12, 24 hours, and eNOS mRNA in PAECs was measured by RT-PCR. After exposed to hypoxia for 24 hours, the expression of eNOS mRNA and protein level increased by 171% +/- 18% (P < 0.05) and 166% +/- 21% (P < 0.01) respectively. These up-regulation effects were prevented by BIM I and Gö6983. Further experiments showed that among 8 isoforms of PKC detected in this study, only nPKCepsilon protein expression was changed in PAECs after exposure to hypoxia for 24 h. After exposure to hypoxia nPKCepsilon was translocated from cytosol to cell membrane, showing the activation of nPKCepsilon during hypoxia. Reporter gene assay showed that hypoxia enhanced eNOS promoter activity up to 2.3 +/- 0.7 fold. In addition, hypoxia did not change the stability of eNOS mRNA. Hypoxia may up-regulate eNOS expression in PAECs by transcriptional mechanism through nPKCepsilon signaling pathway. Higher levels of mRNA observed during hypoxia are due to increased transcription, not to increased stability of mRNA.

Corticotropin-Releasing Factor Receptors Couple to Multiple G-Proteins to Activate Diverse Intracellular Signaling Pathways in Mouse Hippocampus: Role in Neuronal Excitability and Associative Learning

Corticotropin-releasing factor (CRF) exerts a key neuroregulatory control on stress responses in various regions of the mammalian brain, including the hippocampus. Using hippocampal slices, extracts, and whole animals, we investigated the effects of human/rat CRF (h/rCRF) on hippocampal neuronal excitability and hippocampus-dependent learning in two mouse inbred strains, BALB/c and C57BL/6N. Intracellular recordings from slices revealed that application of h/rCRF increased the neuronal activity in both mouse inbred strains. Inhibition of protein kinase C (PKC) by bisindolylmaleimide I (BIS-I) prevented the h/rCRF effect only in hippocampal slices from BALB/c mice but not in slices from C57BL/6N mice. Inhibition of cAMP-dependent protein kinase (PKA) by H-89 abolished the h/rCRF effect in slices from C57BL/6N mice, with no effect in slices from BALB/c mice. Accordingly, h/rCRF elevated PKA activity in hippocampal slices from C57BL/6N mice but increased only PKC activity in the hippocampus of BALB/c mice. These differences in h/rCRF signal transduction were also observed in hippocampal membrane suspensions from both mouse strains. In BALB/c mice, hippocampal CRF receptors coupled to G(q/11) during stimulation by h/rCRF, whereas they coupled to G(s), G(q/11), and G(i) in C57BL/6N mice. As expected on the basis of the slice experiments, h/rCRF improved context-dependent fear conditioning of BALB/c mice in behavioral experiments, and BIS-I prevented this effect. However, although h/rCRF increased neuronal spiking in slices from C57BL/6N mice, it did not enhance conditioned fear. These results indicate that the CRF system activates different intracellular signaling pathways in mouse hippocampus and may have distinct effects on associative learning depending on the mouse strain investigated.

Inhibition of depolarisation-evoked [ 3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by muscarinic (M1) receptors is not mediated by changes in [Ca 2+] i

The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M 1 receptors inhibits K +-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M 1 and M 3 receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium ([Ca 2+] i) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca 2+ i stores to examine the role of protein kinase C and changes in [Ca 2+] i in mediating the inhibition of K +-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K +-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca 2+-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca 2+] i, failed to prevent inhibition of K +-evoked NA release by muscarine. The effect of muscarine on K +-evoked uptake of Ca 2+ e was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca 2+ e-uptake by decreasing the rate at which Ca 2+ entered SH-SY5Y cells via voltage sensitive Ca 2+-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca 2+ from internal stores.

CD19 signal transduction in normal human B cells: linkage to downstream pathways requires phosphatidylinositol 3-kinase, protein kinase C and Ca2+.

CD19 is required for normal antibody responses in mice. We have shown that CD19 enhances the activation of extracellular signal-regulated kinase (ERK) 2 by membrane (m) IgM but otherwise little is known of CD19 signaling in primary human cells. We now ask which pathways link CD19 with ERK2 in human tonsillar B cells. In analyses of signaling intermediates, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin partially suppressed the release of Ca2+ induced by coligation of CD19 and mIgM but the selective PKC inhibitor bisindolylmaleimide I (BIM-I) did not. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, BIM-I and wortmannin each had only a small effect on ERK2 activation induced by surface IgM alone but blocked the enhancement of that activation by CD1 9/mIgM coligation. To analyze the mitogen-activated protein kinase (MAPK) cascade, we measured activation of Raf, MAPK- or ERK kinase (MEK) 1 and ERK2. CD19 consistently enhanced activation of ERK2 and MEK1. However, synergistic activation of Raf was variably observed. In subpopulation analyses, synergistic activation of Raf1 was consistently observed in the IgDlow but not in the IgDhigh cells. Thus, in normal human B cells, PI3K is upstream of the Ca2+ response while PI3K, Ca2+ release and protein kinase C are all required for ERK2 activation, and CD19 enhances the MAPK cascade at multiple levels, depending on the state of differentiation.

Regulation of pharyngeal motility by FMRFamide and related peptides in the medicinal leech, Hirudo medicinalis.

The medicinal leech possesses FMRFamide-like immunoreactivity in neural processes and somata associated with the pharynx and pharyngeal ganglia. The pharynx possessed about 25 immunoreactive somata; about half of the approximately 20 neurons of the pharyngeal ganglia were immunoreactive. We provide brief descriptions of several neurons located in the first neuromere of the subesophageal ganglion involved in controlling pharyngeal motility. Double-labeling experiments indicate that one of these cells, named Swallow neuron 1 (SW1), contains a FMRFamide-like peptide. Stimulation of SW1 caused the mouth to open and the pharynx to dilate. Upon termination of SW1 stimulation, the mouth closed, and a peristaltic wave progressed from the mouth down the length of the pharynx. Stimulation of SW1 did not produce 1:1 postsynaptic potentials in pharyngeal muscle cells. Thus, SW1 is apparently not a motor neuron. The pharynx responded to application of FMRFamide and related peptides by producing a series of 20- to 35-s phasic contractions superimposed upon an increase in basal tonus. The peptide-induced response was quantified by measuring increases in basal tonus, peak tension, and integrated area. Although there were some differences in the order of potency depending upon which parameter was considered, the approximate order of potency of RFamide peptides tested was: pQDPFLRFamide > or = FMRFamide approximately YGGFMRFamide > or = YMRFamide approximately FLRFamide approximately GGKYMRFamide approximately YLRFamide > leucomyosuppressin approximately perisulfakinin. Except for differences in potency, each of the RFamide peptides produced similar contractile waveforms. FMRFamide-induced responses were reduced by the protein kinase C inhibitor bisindolylmaleimide I (10 microM), the nonspecific protein kinase inhibitor H-7 (50 microM), and were increased by the protein phosphatase inhibitor okadaic acid (1 microM). However, the FMRFamide-induced response was unaffected by the protein kinase A inhibitor H-89 (1 microM), the phosphodiesterase inhibitor theophylline (1 mM), the phospholipase A(2) inhibitor OBAA (0.1 microM) or the cation channel blocker amiloride (100 microM).

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells.

The nuclear factor of activated T-cells (NFATp) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-cells. T-cell activation results in the activation of the phosphatase calcineurin (CaN), which leads to the dephosphorylation and subsequent nuclear localization of NFATp. We have investigated the role of kinases in the phosphorylation state and subcellular localization of NFATp. The phosphorylation state and nuclear/cytoplasmic location of NFATp were determined in unstimulated murine HT-2 cells treated with a panel of kinase inhibitors. Two of the seven kinase inhibitors, staurosporine (St) and bisindolylmaleimide I (BI), resulted in the dephosphorylation and nuclear localization of NFATp. These St-induced effects were inhibited by pretreatment with FK506, indicating that CaN activity was required for the observed effects on NFATp. Treatment of cells with ionomycin resulted in NFATp dephosphorylation and nuclear localization. Removal of ionomycin from the cells resulted in the reappearance of phosphorylated NFATp in the cytosol. St and BI also inhibited the re-accumulation of NFATp in the cytoplasm and its re-phosphorylation after ionomycin removal. The re-accumulation of NFATp in the cytosol after ionomycin withdrawal was shown to be energy- and temperature-dependent. Taken together, these results suggest that in unstimulated cells NFATp is actively maintained in the cytoplasm by kinases acting in opposition to basal CaN activity.